Crossing between varieties has been conventionally employed as a method for altering the color of flowers and fruits of plants. However, crossing is carried out between varieties of the same genus, and usually of the same species, and therefore, it is extremely difficult to give specific colors to certain plant species. For example, in spite of longtime efforts of breeders, no one has yet successfully bred a blue rose or a blue carnation.
In recent years, recombinant DNA technology has enabled plant breeding between different species or genus, and it is expected to breed new plants having unprecedented pigment patterns which can not be obtained by the conventional breeding methods by crossing (Plant Molecular Biology, vol.13, p.287-294, 1989). For example, it is reported that petunia showing unprecedented brick-red color on flowers was bred by cloning a gene encoding dihydroflavonol-4-reductase, which is an enzyme participating in pigment biosynthetic pathway, from maize and introducing it into petunia (Japanese Published Unexamined Patent Application No. 2305/90; Nature, vol.330, p.677-678, 1987). Further, a report has been made of a case in which new pigment patterns were produced by introducing the chalcone synthase gene of petunia at the sense or anti-sense orientation to partially inhibit the expression of the gene (Nature, vol.333, p.866-869, 1988; The Plant Cell, vol.2, p.279-289, 1990; The Plant Cell, vol.2, p.291-299, 1990).
Biosynthetic pathways for anthocyanins, which contribute to blue or red color of flowers, have been studied genetically and biochemically in detail using petunia and others (Petunia, Edited by K. C. Sink, Springer Verlag, p.49-76, 1984; The Flavonoids, Edited by J. B. Harborne, Chapman and Hall, p.399-425, 1988; Molecular Approaches to Crop Improvement, Edited by E. S. Denis and D. J. Rewerin, Springer Verlag, p.127-148, 1991). As a result of these studies, it is shown that the presence/absence of hydroxyl group at the 3'- and 5'-positions of the B ring of anthocyanin greatly affects the color of flowers, and also it is shown that, generally, the blue color of flowers is intensified as the B ring is hydroxylated in a higher degree. The hydroxylation of the B ring of anthocyanins occurs at the stage of their precursors, flavanones or dihydroflavonols. As enzymes which catalyze this hydroxylation, two types of enzyme have been known; flavonoid-3'-hydroxylase which hydroxylates only the 3'-position of the B ring, and flavonoid-3',5'-hydroxylase which hydroxylates both the 3'- and 5'-positions. Petunia with blue flowers has both the enzymes, but that with red flowers has only the former one. Plants like roses, carnations, and chrysanthemums do not have anthocyanins which have B ring hydroxylated at both the 3'- and 5'-positions, and therefore are considered not to have the latter type of enzyme.
These hydroxylases are localized in the microsomal membrane and require NADPH as a coenzyme. They are presumed to be members of the cytochrome P450 enzyme group on the basis of their behavior against various inhibitors (The Flavonoids, Edited by J. B. Harborne, Chapman and Hall, p.399-425, 1988; Molecular Approaches to Crop Improvement, Edited by E. S. Denis and D. J. Rewerin, Springer Verlag, p.127-148, 1991).
Cytochrome P450 is an enzyme group which is widely distributed among eucaryotes and procaryotes and which is involved in the biosynthesis of important lipids such as steroids and in the oxidative metabolism of lipophilic substances. In higher animals, it forms a super family consisting of one hundred or more molecular species (J. Biol. Chem., vol.266, p.13469-13472, 1991; Pharmacol. Rev., vol.40, p.243-288, 1988). In plants, cinnamic acid-4-hydroxylase and kaurene oxidase are considered to belong to the cytochrome P450 group (Plant Physiol., vol.96, p.669-674, 1991). Further, a gene encoding a cytochrome P450 enzyme whose function is unknown has been cloned from avocado (Proc. Natl. Acad. Sci. USA, vol.87, p.3904-3908, 1990). As a result of the comparison of the amino acid sequences of various types of cytochrome P450 enzymes, it is known that the sequence of the heme-binding site is conserved (Proc. Natl. Acad. Sci. USA, vol.85, p.7221-7225, 1988; Pharmacol. Rev. vol.40, p.243-288, 1988).
In petunia, flavonoid-3',5'-hydroxylase is encoded by two dominant genes called Hf-1 and Hf-2. The enzymes encoded by the genes are isozymes, and the degree of expression of Hf-1 is higher (Petunia, Edited by K. C. Sink, Springer Verlag, p.49-76, 1984). Further, characteristics of said enzyme of Verbena have been reported (Z. Naturforschung, vol.37c, p.19-23, 1982).
It is also reported that 3',5'-hydroxylase, a key enzyme in the biosynthesis of delphinidin, which is a blue pigment in petunia, has been successfully cloned (Nikkei Biotech, Aug. 26, 1991). However, no report has been made yet of a case in which the cloned gene of said enzyme is allowed to express in a plant to alter pigments in the plant.